This invention relates to vaccines effective in eliciting protective immunity against malaria, in particular vaccines comprising universal T-cell epitopes that elicit T-cell responses in individuals of differing genetic backgrounds.
The public health problems caused by malaria, which currently infects 400-500 million individuals world-wide, have been exacerbated by the emergence of multi-drug resistant parasite strains and insecticide-resistant mosquito vectors. These developments have led to increased efforts to provide an effective vaccine to prevent the mortality and morbidity due to malaria, in particular P. falciparum, the most virulent of the Plasmodial species.
In a mammalian host, malaria infection is initiated by the motile sporozoite stage of the organism, which is injected into the circulation by the bite of infected mosquitoes. The sporozoite is targeted to the host""s liver cells through interaction of a major component of the sporozoite surface membrane, the circumsporozoite (CS) protein, with specific receptors on the hepatocyte surface. Following intracellular multiplication and release from ruptured hepatocytes, the parasites invade red blood cells and initiate the malaria erythrocytic cycle; this phase of infection is responsible for clinical disease and, in the case of P. falciparum, may be lethal.
A major focus of malaria vaccine development has been the CS protein, which is present in both sporozoite and liver stages of the parasite. Polyclonal and monoclonal antibodies specific for an immunodominant B-cell epitope within the repeat region of the CS protein, the (NANP)3 peptide SEQ ID NO:1, neutralize the infectivity of sporozoites of rodent, primate and human malaria species (Nardin et al., J.Exp.Med. 156:20, 1982). Use of the (NANP)3 peptide in a vaccine, however, resulted in only a limited immune response, most probably due to low epitope density and/or lack of a suitable T-cell epitope (Herrington et al., Nature 328:257, 1987).
The present inventors have defined parasite-derived T-cell epitopes using CD4+ T-cell clones derived from four human volunteers immunized by repeated exposure to the bites of irradiated P. falciparum malaria infected mosquitoes. When three of these volunteers were challenged with infective P. falciparum sporozoites, they were protected against malaria, as shown by the total absence of blood stage infection (Herrington et al., Am.J.Trop.Hyg. 45:535, 1991).
Using CD4+ T-cell clones derived from these sporozoite immunized volunteers, two T-cell epitopes have been identified, one located in the repeat region and one in the C-terminus of the P. falciparum CS protein. The T-cell epitope contained in the NH2-terminal repeat region, termed T1, consists of alternating NVDPNANP repeats SEQ ID NO:2 (Nardin et al., Science 246:1603, 1989). The T1 epitope is contiguous to, but antigenically distinct from, the COOH-terminal repeat region which contains the (NANP)3 B cell epitope. The human CD4+ T-cell clones that specifically recognize peptides derived from various combinations of the NH2-terminal repeat region and that contain NVDPNANP do not respond to the (NANP)3 repeat peptide. The T1 repeat epitope is conserved in all P. falciparum isolates sequenced thus far and therefore its inclusion in a vaccine is expected to induce immune responses reactive with parasites of diverse geographical regions.
The second T-cell epitope identified by sporozoite-specific human CD4+ T-cell clones is contained in a peptide spanning amino acid residues numbered 326-345, EYLNKIQNSLSTEWSPCSVT, SEQ ID NO:3 of the P. falciparum NF54 strain CS protein (Moreno et al., Int.Immunol. 3:997, 1991; Moreno et al., J.Immunol. 151:489, 1993). This epitope was shown to be recognized by cytotoxic and non-cytotoxic class II-restricted human CD4+ T-cell clones and class I-restricted CD8+ CTL.
The 326-345 amino acid sequence is unique in that it overlaps both a polymorphic, as well as a conserved region, RII (Dame et al., Science 225:593, 1984), of the CS protein. The conserved RII-plus contains a parasite ligand that interacts with hepatocyte receptors to initiate the intracellular stage of the malaria life cycle. The peptide-specific human CD4+ T-cells recognize a series of epitopes within the 326-345 peptide, all of which overlap the conserved RII found in the CS protein of all Plasmodium species.
The fact that the T* epitope was defined by CD4+ T-cells derived from human volunteers immunized by multiple exposures to the bites of malaria-infected mosquitoes suggests that this peptide sequence is efficiently processed for presentation by HLA class II molecules following exposure to the native CS protein on the sporozoite. It is contemplated that vaccines containing this parasite-derived T-cell epitope can elicit anamnestic responses in naturally-infected individuals and can provide for vaccine-induced immunity to be maintained by continued exposure to the parasite under natural conditions.
Class II-restricted CD4+ T-cells play a central role in the induction of both cellular and humoral immunity to the pre-erythrocytic stages of the malaria parasite (Nardin et al., Ann.Rev.Immunol. 11:687, 1993). If the T-cell epitopes contained within a synthetic malaria vaccine bind to only a limited range of class II molecules, the vaccine may fail to elicit immune responses in individuals of diverse genetic backgrounds. Earlier studies have shown that the (NANP) repeats of the P. falciparum CS protein induced low or undetectable T-cell responses in naturally-infected individuals living in malaria endemic areas (Herrington et al., Nature 328:257, 1987; Etlinger et al., J.Immunol. 140:626, 1988; Good et al., Proc.Natl.Acad. Sci. USA 85:1199, 1988).
Thus, there is a need in the art for parasite-derived T-cell epitopes that bind to most, if not all, class II molecules for inclusion in immunogenic compositions and vaccines, to provide protective immunity against malaria in individuals of diverse genetic backgrounds.